The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A (PKA)-activated chloride channel that is found on the apical surface of a number of cell types of epithelia. One unusual feature of this protein is that during biogenesis, as much as 75% of CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway, suggesting that CFTR is intrinsically unstable. DeltaF508, the most common mutation in CFTR, results from a folding defect and is completely degraded by ERAD. Previous studies on CFTR maturation utilized heterologous, over-expression systems because of the limited amounts of CFTR that are synthesized in epithelia. Our preliminary data in an airway epithelial cell line, Calu-3, suggest that all of the newly synthesized wild-type CFTR is converted to the maturely glycosylated form of this protein. These data question whether wild-type CFTR is unstable and allow us to pose the following hypothesis: endogenous expression of wild-type CFTR is slow, but efficient in epithelial cells. Given the over-estimation of the degradation of the wild-type protein by ERAD, we wonder if this might also be true for the deltaF508. Additionally, deltaF508 CFTR can be released to the surface after low temperature treatment, and recent studies report that some deltaF508 is expressed on the surface of certain epithelia. Based on this, we hypothesize that in certain epithelia some deltaF508 escapes ERAD and is delivered to the apical surface. To test these hypotheses, we propose the following specific aims: (1) to determine the efficiency, transport kinetics, and pathway of wild-type CFTR biogenesis in polarized epithelia; and (2) to test the hypothesis that there are cell-type specific differences in deltaF508 CFTR maturation and delivery to the cell surface. To complete these aims, we will utilize airway, pancreatic, and colonic/intestinal epithelia under polarized conditions to follow wild type and deltaF508 maturation and surface delivery. We will monitor maturation and protein half-life using metabolic pulse-chase experiments, follow the fate of CFTR through the secretory pathway using both biochemical and morphological approaches, and follow the surface stability and function using a sensitive surface biotinylation assay and Ussing chamber analysis. Completion of these experiments will provide novel and important information on a physiologically important, multi-domain; integral membrane protein is synthesized in a number of epithelial cell types.